Main Conference - Day 3 (May 17)Friday, 17 May 2024

Chemically modified oligonucleotides that modulate RNA hold great promise for the treatment of human disease. Wave Life Sciences is advancing new chemistries to generate stereopure, chimeric backbone-containing oligonucleotides—those in which the chirality of each backbone linkage has been precisely controlled during chemical synthesis—with the aim of improving their drug-like properties. We will provide an overview of the methods we have developed to synthesize, manufacture, and quality control stereopure chimeric oligonucleotides containing PN (phosphoryl guanidine) backbone linkages in combination with more traditional phosphodiester (PO) and phosphorothioate (PS) backbone linkages. We will describe how stereochemical stability and stereochemical identity are established and interrogated, using multiple analytical techniques, to support the manufacture of stereopure oligonucleotides for clinical use.

During development of analytical methods for release and stability testing of siRNA compounds, attention should be paid to shifts in equilibria between intended duplex, single strands, duplex impurities, and single strand impurities resulting from the analysis itself. Mass spec-compatible chromatographic conditions enabling detection of impurities co-eluting with parent and other oligonucleotide impurities for GalNAc- and peptide-conjugated siRNA duplexes will be presented. T_m by UV and DSC measurements were used to assess if LC method conditions were re-naturing single strands dissociated in samples.

Radionuclide Theranostics - thanks to the recent approvals of Lutathera and Pluvicto – have gained significant momentum. A number of new theranostic approaches are currently translated into the clinic including new targets, new binders, new radionuclides, synergistic combination treatment and radiosensitizers. Overall the field of radionuclide theranostics involves a number of moving targets. This presentation aims to provide a snapshot of the currently most promising radionuclide theranostics on the brink of being clinically translated.

Integrin 𝜶_vβ₃ plays an essential role in regulating angiogenesis, a key process in tumor growth. Restricted over expression of integrin 𝜶_vβ₃ on the activated endothelial cells of neo-vasculature of tumor makes 𝜶_vβ₃ an invaluable molecular target that may help in early diagnosis and management of various solid tumors. The study aims to describe bench to bedside development of radiolabeled RGD peptide targeting 𝜶_vβ₃ integrin.

Bicycle® molecules are a novel peptide-based modality consisting of constrained peptides that form a bi-cyclic structure via ligation to a chemical scaffold and which are discovered using the Bicycle® phage display platform and have potential broad utility, allowing efficient and targeted delivery of different classes of payloads into tumors. We are developing Bicycle Radionuclide Conjugates (BRC™ molecules), in which Bicycle® molecules are employed as targeting vectors to deliver radioisotopes to tumors for cancer imaging and therapy. Bicycle® molecules exhibit properties that make them an ideal modality for radionuclide delivery. They can achieve exquisite binding specificity and high binding affinity and have demonstrated rapid tumor penetration, resulting in high accumulation of payload in the tumor but with limited exposure to normal tissues. We have used in vitro cell binding assays and mouse cell line derived xenograft models to characterize early MT1 targeted BRC™ molcules to establish binding properties and in vivo biodistribution and demonstrated that BRC™ molecules can be chemically optimized to improve their in vivo biodistribution profiles.

Intellia is a leading clinical-stage gene editing company focused on the development of CRISPR-based therapies. Interim clinical data with NTLA-2001, an investigational in vivo CRISPR-based therapy with the potential to be the first single-dose treatment for ATTR amyloidosis, will be presented.

Beam Therapeutics is a leading clinical stage company focused on next generation gene editing therapies. Beam’s proprietary base editing technologies are designed to enable a new class of precision genetic medicines that target a single base in the genome without making a double-stranded break in the DNA. This approach aims to create a more precise and efficient edit compared to traditional gene editing methods, which operate by creating targeted double-stranded breaks in the DNA, resulting in potential challenges associated with unwanted DNA modifications. An overview of Base Editing as well as its diverse therapeutic applications will be presented.

Adipose tissue is a critical endocrine organ for energy and hormonal homeostasis, and disruption of these highly regulated processes can lead to the induction of metabolic disease such as obesity and type 2 diabetes. There remain significant challenges, however, in the development of novel therapeutics capable of correcting these processes in adipose tissue. To address adipose-related disease, Arrowhead has developed a TRiM™ platform for the delivery of RNAi therapeutic candidates to white adipose tissue. This platform provides a novel and highly efficient mode of delivery of siRNA, allowing for low and infrequent dosing regimens via subcutaneous administration. The TRiM™ adipose platform has achieved notable gene knockdown (≥ 90%) and long duration of effect in both rodent and non-human primates. The TRiM™ adipose delivery platform may help address the unmet need for novel therapeutics capable of treating adipose-related diseases and disorders.

Phosphorodiamidate morpholino oligomers (PMOs) are short single-stranded oligonucleotides comprising a backbone of morpholine rings connected by phosphorodiamidate linkages. The manufacture of PMO drug substances involves solid-phase oligomer synthesis and subsequent cleavage/deprotection followed by purification and lyophilization. During the manufacturing process, a variety of impurities are generated from various sources. These process-related impurities are often structurally related to their parent PMO. Determination of impurity profile of drugs to confirm quality and thereby ensure safety and efficacy is essential. Herein, we present the development of a robust 2D-LC/MS workflow and its application for impurity profiling of PMOs. PMOs and the impurities were separated by two-dimensional LC with orthogonal modes of separation and detected by Quadrupole Time-of-Flight mass spectrometry. The developed 2D-LC/MS workflow was successfully applied to the impurity profiling of various PMOs with different sequences and lengths.

Resolution performance of LC and MS techniques is challenged by development of long therapeutic oligonucleotides such sgRNA. We will discuss the application of modern method of ultra-performance LC for separation and MS characterization of long oligonucleotides and other classes of nucleic acids.

Prime editing is a “search-and-replace” gene editing technology that can correct disease-causing genetic mutations at their precise location in the genome, without requiring double-strand DNA breakage. Prime editing offers a potential therapeutic platform for a broad range of challenging diseases. Comprehensive analytical methods are being developed to assess the quality of gene editing critical raw materials and drug substances (e.g., prime editing mRNA or protein, nicking guide (NG)RNA, prime editing guide (PEG)RNA), because of the potential effects quality can have on, for example, gene editing accuracy and efficiency. PEGRNAs can be particularly challenging to analyze because of their length, secondary structures, and complex activities. A series of HPLC- and mass spectrometry-based analytical methods were developed to assess PEGRNA purity, stability, mass, impurity ID/quantitation, and biochemical activity.

The recent approvals of Lutathera and Pluvicto have highlighted the potential of Radiopharmaceutical Therapy (RPT) as a secure and efficient targeted modality for treating various solid tumors. The successful development of RPT necessitates methodical optimization and a thorough evaluation of the targeting moiety, linker, chelator, and the selection of radioisotopes. RayzeBio is at the forefront of innovation in this domain, employing a data-driven drug discovery approach to systematically identify optimal RPT agents against clinically validated oncology targets that have yet to be addressed using RPT. In this presentation, we will share the application of this approach to develop and optimization of potential RPT agents for the treatment of GPC3-expressing tumors.

Aktis Oncology is developing radiopharmaceuticals based on miniprotein binders. Miniproteins have ideal properties for radioconjugates being highly selective and potent with excellent tumor penetration properties while also rapidly clearing from the periphery via kidney filtration sparing healthy tissue. Aktis has generated multiple first-in-class programs using the miniprotein platform demonstrating its broad utility.

Radioligand therapy (RLT) has recently demonstrated attractive clinical benefits. Such early success has promoted the race for next wave RLTs where exciting opportunities and unique challenges have been both presented. Here we will disclose the discovery of ²²⁵Ac-FL-020, a novel PSMA-targeting RLT candidate identified by our proprietary Clear-X technology platform.

Editas Medicine will present clinical data for the RUBY and EdiTHAL trials. In both trials, observed pharmacodynamic responses and preliminary efficacy data confirm proof of concept for reni-cel (EDIT-301) mechanism of action. Reni-cel was well-tolerated and demonstrated a safety profile consistent with myeloablative conditioning with busulfan and autologous hematopoietic stem cell transplantation in all treated patients in the two trials (n=17).

Understanding the role of key cellular mediators is key to developing the next generation of safe, well tolerated non-viral delivery systems. We will discuss advances in tuning the degradation rate, immune activation, and tissue targeting of biodegradable COATSOME ® SS Series for development of distinct LNPs for cell therapy, gene editing, and vaccines.

The success of the Covid vaccine has validated mRNA as a powerful vaccine technology. Owing to its ease of manufacturing and fast design and development cycle, more mRNAs have entered into clinical phases. However mRNA vaccine for veterinary use remains a less explored area. Here we present a veterinary mRNA development platform that can accelerate animal vaccine development and make low-cost mRNA vaccine a reality.

mRNA is a biotherapeutic modality that has been successfully used in vaccines and vaccine development. Because of the fast-tracked success, high demand for robust analytical methods used to make critical quality decisions are needed. The manufacturing process of mRNA molecules consists of in vitro transcription reactions in which the desired final product is over 1000 nucleotides in length, has a 5’ Cap and a Poly (A) tail. Both the 5’ Cap and the Poly (A) tail help to stabilize the RNA molecule and improve its translation. Contaminants, such as degradation products (incomplete capping and shorter than expected Poly(A) tails), can still be present in the final product and need to be quantified to assess mRNA purity. This study shows an LC-MS characterization method for three mRNA key critical quality attributes: 5’ Cap, Open Reading Frame (ORF) and Poly (A) tail.

The solid phase oligonucleotide synthesis based on sequential coupling of phosphoramidite monomers is a well-established industrial manufacturing process, currently performed routinely on kilo scale mainly due to limitations of synthesis and purification processes. Novel approaches towards more efficient, scalable, and sustainable large-scale manufacture will be discussed supporting future commercialization of the expanding range of high-volume siRNA therapeutics.

The use of ultrafiltration/diafiltration to process antisense oligonucleotides will be examined including its capabilities and limitations, membrane properties, sieving coefficients, along with concentrations, flux, and yields achieved. Additionally, effects of buffer types, permeability, viscosities, and impact on clearance will be presented.

Oligonucleotide impurities associated with the installation of mesyl phosphoramidite internucleotide linkages during solid-phase synthesis have been identified and characterized. The impurities result from modification of guanosine residues. In this presentation, we will discuss the impurities’ structures and mechanisms of formation as well as effective mitigation strategies to limit their formation.

Patients greatly prefer the oral route of administration for pharmaceuticals. Limited oral absorption of biologics (peptides, proteins, RNAs, and antibodies) is a major challenge. The BIONDD^TM capsule delivers biologic drugs with a bioavailability like SC injection creating a broad platform for oral delivery of drugs that would be injected today.

We present a comprehensive peptidomimetic SAR approach, combined with cellular target engagement assays to improve the current VHL ligand. We identified the 1,2,3-triazole group as an optimal substitute for the amide bond, and incorporated conformationally constrained alterations on the right-hand side, led to picomolar binding affinity and improved oral bioavailability.

CRISPR-Cas technologies offer precise genome manipulation for gene therapy. Parasutterella secunda Cas9 is a high-fidelity enzyme capable of gene editing in mice. We report the Cryo-EM structure of PsCas9 and engineered it and its sgRNA. The engineered variant, ePsCas9, maintains high-fidelity and shows superior gene editing in mouse liver, outperforming SpCas9 with no safety concerns. ePsCas9 is a highly efficient and precise tool for in vivo gene editing.

Our lab has developed new molecular technologies for genome editing and cell engineering, including PASTE for large DNA integration, RNA guided CRISPR proteases, and a novel technology for programmable mRNA therapies. These advances enable precise genome editing and cell state manipulation, with significant implications for therapeutics and diagnostics.

Reverse transcriptases, used in prime editing systems, exhibit lower fidelity, processivity and dNTP affinity than many DNA-dependent DNA polymerases. I will present a DNA-dependent DNA polymerase (phi29), untethered from Cas9, enables efficient editing from a synthetic, end-stabilized DNA-containing template in human cells. Compared to prime editing, DNA polymerase editing avoids autoinhibitory intramolecular base pairing of the template, facilitates template synthesis and supports larger insertions.

The 2’-NMA chemistry employed in the synthesis of antisense oligonucleotides (ASOs) introduces distinctive synthetic challenges characterized by high branchmer levels and a low purity profile. The intricacies of branchmer formation were studied through targeted syntheses and high-resolution mass spectrometry (HRMS) analysis. It was observed that the branchmer formation can be effectively suppressed by adjusting the process parameters.

Poor real-world medication adherence prevents patients from receiving the full potential benefits of their treatment, contributing to 125,000 annually avoidable deaths and over $100B in avoidable healthcare costs in the US alone. To address this challenge, Vivani Medical is developing a miniaturized long-term subdermal implant to guarantee medication adherence over many months. The first application is a twice-yearly exenatide (GLP-1) implant under development for the treatment of Type 2 Diabetes and Obesity.

I-PGI combines the specificity of CRISPR/Cas9 with proprietary integrases that allow for the insertion of any DNA sequence of any size into a specific programmed location. We will share our progress developing this technology for both in vivo (integrative gene therapy) and ex vivo (cell therapy) applications.